Microspheres for use in the treatment of cancer

ABSTRACT

The subject invention pertains to novel materials and methods for use in treating patients afflicted with oncological disorders, such as tumors. One aspect of the invention concerns methods for treating tumors comprising the administration of drug loaded pH-sensitive microspheres to a patient in need of anticancer therapy. The pH-sensitive microspheres have a swelling transition with the pH range found in or near tumor tissue. When the microspheres swell, the loaded drug is released into the microenvironment of the tumor tissue. The subject invention also pertains to novel microspheres useful in the methods of the invention. The microspheres are capable of effectively releasing a loaded substance at a pre-determined pH. The subject invention also concerns novel methods for synthesizing the microspheres of the present invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/143,774, filed Aug. 31, 1998, now abandoned.

BACKGROUND OF THE INVENTION

With advances in antibiotics and vaccines there has been a reduction inthe seriousness of many infectious diseases; however, cancer stillremains as a mostly incurable threat. In fact, cancer accounts for about10 percent of all deaths in the U.S. every year (Oppenheimer, 1985). Oneobstacle in the treatment of cancer is that the basic mechanism ofcancer development and propagation is not well understood and,therefore, investigation into possible cancer treatments may requireknowledge from a variety of different disciplines (Braun, 1974, Muir,1988). Cancer patients must withstand the debilitating mental andphysical effects throughout the long duration of the disease which alsoresults in an economic burden to both the patient and the community(Busch, 1974).

The mortality rate for patients diagnosed with either primary orsecondary liver cancer is very high. Many new approaches towardspossible treatments are currently being investigated; however, successeshave been minimal and surgery still remains as the best form oftreatment, even though less than 10 percent of the patients are suitablefor this option (Kemeny et al., 1995). Non-surgical forms of treatmentinclude various routes of chemotherapy in which toxic chemotherapeuticdrugs are delivered to the liver tumors, either systemically (throughoutthe entire body) or regionally (directly into the liver). Thechemotherapeutic drugs such as fluorodeoxyuridine (FUDR) and doxorubicin(adriamyacin) work by having a greater toxic effect on actively dividingcells such as cancer cells, rather than most normal tissues. The goal inthis form of treatment is to deliver a high dose of the drug to thetumor tissue while keeping the concentration of the drug (and its toxiceffects) in normal tissue to a minimum. The toxic side effects of thechemotherapeutic agents may be the limiting factor in determining thedrug concentration delivered to the patient. In many cases there isinsufficient killing of the tumor cells and regrowth and spreading mayoccur (Bhattacharya et al., 1994). In addition, with conventionalsystemic or regional treatment, the excess drug which does not contacttumor tissue degrades the condition of the healthy tissue and,therefore, can become the limiting factor in dose concentration (Kemenyet al., supra). An ideal situation would occur if the toxic effects ofthe drugs could be completely localized within the liver tumor tissuewithout affecting the surrounding healthy tissue, enabling a higher drugconcentration to completely kill all of the cancer cells.

Chemotherapy is often combined with another form of treatment termedembolization in which the blood supply to the tumor is essentiallyreduced or stopped either temporarily or permanently in an attempt toarrest the tumor growth or cause regression. Typical embolic agentsinclude steel coils as well as polyvinyl alcohol sponge (IVALON),collagen, gelatin sponge (GELFOAM), albumin, and starch materials thatmay be in the form of microspheres or particles. Because healthy livertissue has a dual blood supply through the hepatic artery and the portalvein, and most hepatic tumors are oxygenated almost exclusively from thehepatic artery, the theory behind embolization is that this artery canbe obstructed by injections of these materials in an attempt to starvethe tumor of its blood supply without injury to the majority of theliver. If this technique is used in combination with regionalchemotherapy the drug can be contained within the tumor tissue forlonger periods of exposure time (Lin et al., 1988, Kemeny et al., 1995).In many cases, however, a collateral circulation will appear andcircumvent the blockage or the embolized artery will reopen allowingblood to once again feed the tumor. If viable tumor cells still remain,this will allow them to regrow tumor tissue (Kemeny et al., 1995).

Natural and synthetic polymers have been used to produce microspheresfor a variety of biomedical applications including general and targeteddrug delivery devices. The term microsphere generally refers tospherical particles between 2 nm to 50 nm in diameter but smaller sizes(usually below 1 micrometer) may be referred to as nanospheres. Microparticles are similar but usually irregular in shape (Arshady, 1993). Asmentioned previously, some polymeric spheres and particles have beenused as embolic agents for the treatment of liver cancer. Thesematerials, such as starch, poly (vinyl alcohol), and gelatin, do notrelease a drug but rather serve to occlude the blood flow after a drughas already been delivered in order to allow increased retention timewithin the liver (Lin et al., 1988). Work has also been done in thedevelopment of polymeric microspheres that deliver anticancer drugs in acontrolled fashion. For example, a feasibility study was done for theoral delivery of an anticancer drug, methotrexate, encapsulated indegradable gelatin microspheres. The microspheres were coated with thenatural polymers chitosan and alginate which would enable themicrospheres to pass through the gastrointestinal tract to reach theintestine where the drug action or absorption is desired. In theory,higher concentrations of the toxic drug could be delivered using thistargeted delivery system rather than systemic treatment while reducingside effects which include vomiting, diarrhea, gastro intestinalulceration, and liver and kidney damage (Narayani et al., 1995).Experiments by Kato et al. (1981) showed that mitomycin C or cisplatincould be encapsulated within biodegradable ethyl-cellulose microcapsulesfor possible use in chemoembolization, and a separate study showed thatcisplatin could be loaded into poly(lactide) microspheres such thatcontinuous release could be obtained for a period of several days to aweek. Cisplatin is one of the most potent chemotherapeutic agents knownand is commonly used to treat liver tumors. Since the drug can causemany toxic side effects, the use of microspheres has been suggested totarget its action by hepatic arterial injection and controlled release(Spenlehauer et al., 1986). This idea is supported by a separate studyusing a rat model that showed microspheres of a certain size rangedelivered to the liver via the hepatic artery were found to beconcentrated in a 3:1 ration of tumor tissue to liver tissue forimplanted salivary adenocarcinomas (Meade et al., 1987). While thesedegradable microsphere systems would be able to achieve continuousrelease within the liver, they remain non-tumor specific and drugconcentrations would ultimately be limited by the toxic side effectsproduced, including damage to healthy liver tissue (Kemeny et al.,1995). One current area of research that attempts to increase thetargeting of anticancer treatment is with the use of magneticallydirected microspheres. Hafeli et al. (1994) have developed poly(lacticacid) microspheres which can be loaded with Yttrium-90 and incorporatedwith magnetite such that it may be possible to magnetically direct theradiotoxic effect of the spheres to be more concentrated near tumorsites.

Malignant cells show an increased rate of glucose uptake and aerobicglycolysis with the resulting formation of lactic acid (Volk et al.,1993). In normal cells the uptake of glucose is accomplished by membraneproteins known as glucose transporters. Depending on the cell type, theproteins show different patterns of expression, hormone responsiveness,and transport properties. When cells transform into the malignant statethe number of the glucose transporter proteins per cell is commonlyincreased. Because of this, the uptake of glucose into malignant cellsis no longer regulated by systemic or cellular demands, and is insteadcontrolled almost completely by the extracellular concentrations (Jahdeand Rajewsky, 1982). This means that the increased amounts of lacticacid produced by the aerobic metabolism can be further increased by thesystemic infusion of glucose, resulting in local tumor pH values thatare lower than that for healthy tissue (which remains consistently closeto 7.4) (Volk et al., 1993).

As can be understood from the above, there remains a need for a drugdelivery system for cancer treatment, such as primary or secondary livercancer, that would release an anticancer agent in high concentrationsonly within the tumor tissue while healthy tissue would remainrelatively unaffected.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to novel materials and methods for use intreating patients afflicted with malignancies. Specifically exemplifiedis a method of treating hepatic tumors comprising the use of drug loadedpH-sensitive microspheres. In one embodiment, pH-sensitive microspheresof the invention exhibit a swelling transition within the pH rangetypically found in tumor tissue. The materials and methods of thesubject invention provide a novel treatment of cancer which specificallytargets tumor tissue and reduces the damage to surrounding healthytissue. Further, the subject invention provides a viable alternative tosurgical techniques, in addition to reducing the amount of adverse sideeffects such as vomiting, myelosuppression, cardiac toxicity, pulmonaryfibrosis, hepatobiliary toxicity, and pericholangitis commonlyassociated with other current non-invasive treatments.

One aspect of the subject invention is directed towards methods oftreating a tumor comprising administering an effective amount ofmicrospheres that are capable of releasing a substance at apre-specified pH. The substance contained in the microspheres caninclude, but is not limited to, cytotoxic agents, chemotherapeuticagents, and radionuclides.

The subject invention also pertains to novel microspheres that can beloaded with a substance useful in treating cancerous cells. Themicrospheres are capable of effectively releasing the loaded substanceat a pre-determined pH. The microspheres can be designed to releasetheir substance over a period of time at a pH that is typically found inor near cancerous tissue.

The subject invention also concerns methods for preparing microspheresof the present invention. The methods of the invention allow for thepreparation of microspheres whereby the amount of a selected substanceto be loaded in a microsphere, as well as the release characteristics ofthe microspheres, e.g., release/time curve and pH, can all be selectedfor and manipulated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 show the release of the test dye over a 14 day period at pH6.0, 6.5, and 6.8 for the 67/30/3, 65/34/1, 60/38.5/1.5 ethylmethacrylate (EMA)/(diethyl amino)ethyl methacrylate (DEA)/divinylbenzene (DVB) microsphere compositions. In FIG. 1, the microspheresshowed complete release at a pH of 6.0 after 13 days, and less than 50%release at pH 6.5 and 6.8 after the full 14 days. FIGS. 2 and 3 showthat by increasing the DEA content (as well as decreasing the amount ofcrosslinking) of the spheres, an increase in the overall releasekinetics results.

FIG. 4 shows dye release for the 50/49/1 composition at pH's of 6.5,6.8, 6.9, and 7.4.

FIG. 5 shows the release curves for the FUDR in buffer at 4 different pHvalues.

FIG. 6 shows doxorubicin release from 50/49/1 EMA/DEA/DVB microspheres.

FIGS. 7 and 8 show release profiles for three different size microspheresamples at two different pH values. In FIG. 7, the curves virtuallyoverlap which shows that at pH 6.5 the microsphere swelling is such thatthe dye release has little or no dependence on the sphere size. However,in FIG. 8, which shows release at pH 6.7, the 80-150 μm diameter spheresshow a noticeably slower release with a 14 day total of only 86%compared to 98% for the smaller spheres.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention pertains to novel methods and compositions fortreatment of cancer. In one embodiment, the invention provides methodsof treatment utilizing pH-dependent microspheres which release a drug orother therapeutic agent within a predetermined pH range. Themicrospheres are prepared so that the therapeutic agent is released nearcancer or tumor cells due to the lower pH within the microenvironmentsurrounding those cells. The methods and materials of the subjectinvention can also be used to treat other diseases where pH of themicroenvironment in and around the diseased tissue or cells is lowerthan in non-diseased tissue or cells.

The subject invention utilizes a pH-sensitive, non-degradable carrier(microsphere) having a matrix that can swell as a result of the lower pHassociated with the physiological microenvironment of, for example, atumor resulting in the gradual release of a selected substance containedwithin the microsphere, such as a chemotherapeutic agent. In thismanner, release of the substance from the microsphere is localizedwithin the tumor tissue, with minimal release occurring elsewhere in thesurrounding tissue where the pH is at normal levels. In one embodiment,depending on the tumor pH, glucose can be infused to create a furtherincrease in the H⁺ ion activity of the tumor tissue, resulting in anincreased release rate of the agent from the swollen microsphere matrix.The microspheres can be injected via a blood vessel proximate to thetumor, e.g., the hepatic artery in liver tumors, such that injectedmicrospheres are lodged preferentially within the tumor tissue. Thisallows a drug concentration to be released that is substantially greaterthan that delivered by other techniques without the associated highlevel of toxicity affecting the healthy tissue. In one embodiment,microspheres are designed to release substantially all of their contentsover about a two week period at a pH range of about 6.7 to 6.8.

The subject invention pertains to a method of treating cancer byadministering an effective amount of microspheres containing a selectedsubstance or substances, wherein the microspheres can release thesubstance(s) at a selected pH. Substances for use with the microspheresof the present invention include, but are not limited to, cytotoxicagents, chemotherapeutic agents and radionuclides. In a specificembodiment, administration of an effective amount of microspheresincludes the injection of the microspheres in a blood vessel, such as anartery, proximate or upstream of a metastasis, or tumor. Microspherescan be loaded with a selected substance or substances by contact with asolution containing the substances. Loading can also include the stepsof prewashing the microspheres with an organic solvent, contacting themicrospheres with a solution containing the substance(s), and washingthe loaded microspheres with water. The microspheres can be loaded byother suitable methods known to those skilled in the art.

A preferred embodiment of the subject invention pertains to a method oftreating a tumor comprising preparing pH-dependent microspheres whichcontain a selected anti-tumor substance and injecting the microspheresin a blood vessel suitable for carrying the microspheres to the tumor.

Another aspect of the subject invention pertains to novel non-degradablemicrosphere compositions useful for pH-regulated release of therapeuticagents, such as anti-neoplastic drugs. The microspheres can be loadedunder conditions where a substance is incorporated within the matrix.The loaded microspheres can then release the substance when exposed to acertain pH range. In one embodiment, the microspheres of the subjectinvention are designed to release the substance contained within themicrospheres over a period of time when exposed to a pH typicallyassociated in the microenvironment of cancerous tissue. Preferably, themicrospheres are loaded with any substance suitable to treat cancercells such as cytotoxic agents, chemotherapeutic agents, andradionuclides. The microspheres of the subject invention can be made ofany materials known by those skilled in the art that can swell andcollapse under particular conditions, e.g., cross-linked polymer gelsthat possess either weakly acidic or basic pendant groups. Preferably,the matrix of the gels is composed of a co-polymer containing ahydrophobic component and a less hydrophobic ionizable component whichare then lightly cross-linked.

The composition of the subject microspheres can comprise one or more ofthe following: poly(n-alkyl methacrylate-co-diethylaminoethylmethacrylate); poly(sterene-co-vinyl pyridine), “PSVP”; divinylbenzene,“DVB”; hydrophobic N-alkyl methacrylate, such as n-butyl methacrylate;(dimethylamino)ethyl methacrylate, “DMA”; diethyl(aminoethyl)methacrylate, “DEA”; ethyl methacrylate, “EMA”; poly(vinyl pyrrolidone),“PVP”; poly(ethylmethacrylate), “PEMA”; and poly(methylmethacrylate),“PMMA.” In one embodiment, the microspheres of the subject inventioncomprise ethyl methacrylate, diethylaminoethyl methacrylate, and divinylbenzene. Preferably, the mole percent ratios of ethyl methacrylate,diethylaminoethyl methacrylate, and divinyl benzene are about 65 toabout 50, about 34 to about 49, and about 1 to about 3, respectively.Most preferably, the microspheres of the subject invention have molepercent ratios of ethyl methacrylate, diethylaminoethyl methacrylate,and divinyl benzene of about 50:49:1, respectively.

The present invention also concerns methods for preparing microspheresof the subject invention. In one embodiment, the method comprisespreparing a monomer solution comprising ethyl methacrylate,diethylaminoethyl methacrylate, and divinyl benzene; mixing the monomersolution; and heating the monomer solution. In a specific embodiment,the monomer solution is prepared by mixing ethyl methacrylate,diethylaminoethyl methacrylate, and divinyl benzene having mole weightratios of about 50 to about 65, about 34 to about 49, and about 1 toabout 1.5, respectively, with recrystallized azobisisobutyronitrile(AIBN). In a preferred embodiment, the method for preparing microspherescan further comprise cleaning and drying the microspheres.

A specific embodiment of the subject invention pertains to microspheresuseful in the medical treatment of a disease, where the microspheres arecapable of being loaded with a substance and then effectively releasingthe incorporated substance when exposed to a predetermined pH.Preferably, the microspheres of the subject invention are constructed soas to release the loaded substance at a pH of from about 6.0 to about7.4. More preferably, the release pH can be from about 6.3 to about 7.0.Most preferably, the release pH can be from about 6.5 to about 7.0. Asused herein, the term “effectively releasing” is intended to mean anextended release at the selected pH suitable for treating a tumor. Inone embodiment, the microspheres are capable of effectively releasingabout 50% of their loaded substance after 14 days at a pH of about 6.8.The microspheres can have a loading capacity of up to about 20% of theirweight. In one embodiment, the microspheres of the subject inventionhave a weight percentage of loading of up to about 16.6%.

The microspheres can be constructed in sizes suitable for particularapplications. The size is typically selected as a size that is smallenough to allow the microsphere to be located close to the diseased orcancerous cells or tissue but not so small that the microspheres willpass through the circulatory system to other areas of the body. In oneembodiment, the microspheres of the subject invention range in size fromabout 10 μm to about 150 μm in diameter. In a preferred embodiment, themicrospheres are from about 25 μm to about 35 μm in diameter.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Microsphere Production: Suspension Polymerization Technique

Crosslinked polymer gels (hydrogels) that possess either weakly acidicor basic pendant groups will expand in a solvent, dependent on the pHand ionic composition of the solution. Acidic groups cause the gel toexpand as the pH of the solution increases, whereas basic groups resultin an expansion as the pH decreases. It has been found that forhydrophobic gels in an aqueous environment, the gel will remain in acollapsed state until the pH reaches a critical value upon which theprocess of gel ionization is initiated causing an abrupt increase inequilibrium swelling. The equilibrium swelling of these types of polymergel networks is determined by a balance of the following threeforces: 1) the free energy of mixing associated with the polymer gelmatrix and the solvent, 2) the net osmotic pressure within the matrixdue to the mobile counter ions in the solvent surrounding the fixedcharged groups of the polymer gel, and 3) the elastic response of thepolymer matrix (Siegel et al., 1988). Some examples of pH sensitive gelmaterials include cellulose (Grignon et al., 1980),poly(acrylamide-co-acrylic acid) (Peppas et al., 1991),poly(styrene-co-vinyl pyridine) (Batich et al., 1993), and poly(n-alkylmethacrylate-co-diethylaminoethyl methacrylate) (Siegel et al., 1988).The gel matrix is usually composed of a copolymer containing ahydrophobic component and a less hydrophobic ionizable component whichare then lightly crosslinked.

Spherical copolymer microspheres were produced using a suspensionpolymerization technique. A stabilizer solution was first made using 5 ghydroxyethyl cellulose (Aldrich), 1.25 g bentonite (Fisher Scientific),100 g NaCl (Aldrich), and roughly 525 ml distilled water. The solutionwas stirred at 60-70° C. for one hour and then filtered through a 53 μmsieve (Fisher Scientific) into a reaction vessel fitted with amechanical stirrer (RW 20 DZM Janke & Kunkei Ika-Werk, Germany). Themonomer solution (60 g total) was prepared by mixing different ratios ofethyl methacrylate (EMA)/(diethyl amino)ethyl methacrylate (DEA)/divinylbenzene (DVB) (Aldrich/Aldrich/Monomer-Polymer Laboratories) with 250 mgof recrystallized AIBN (Aldrich) (0.4 wt % by monomer). The monomerswere vacuum distilled and the divinylbenzene was washed with a 10%sodium hydroxide aqueous solution prior to use. Argon was bubbledthrough the monomer solution for 1-2 minutes prior to the solution beingadded to the reaction mixture. The complete reaction solution wasstirred at 900 rpm for the entire process. The heating profile wascarried out using a heating band around the reaction vessel that wascontrolled using a Honeywell IACD (mini-pro) temperature profilecontroller. The temperature profile was as follows: 1) 45 minute rampfrom 30 to 75° C., 2) hold at 75° C. for 30 minutes, 3) 30 minute rampfrom 75 to 85° C., 4) hold for 4 hours at 85° C. then shut off. Aftercooling to room temperature the reaction vessel was disassembled and thecontents were poured into a 2 liter graduated cylinder that was thentopped off with water. The microspheres were initially cleaned by aprocess of repeated sedimentation and then re-suspension in water overthe period of a week. The washed spheres were collected, rinsed withmethanol (Fisher Scientific), dried in a paper cone filter (Whatman) for12 hours, and then placed in a vacuum oven (Napco, E-Series Model 5831)at 60° C. for an additional 12 hours to remove all water and methanol.The dried spheres were broken up with a spatula and sieved to obtainsamples with diameters of less than 35 μm, 35-50 μm, 50-80 μm, and80-150 μm. A total of four trial compositions were produced with67/30/3, 65/34/1, 60/38.5/1.5, and 50/49/1 mole percent ratios ofEMA/DEA/DVB.

EXAMPLE 2 Microsphere Loading and Release of Dye

All four compositions produced in Example 1 were tested for releaseproperties using 9-aminoacridine (Aldrich) (free from HCl) as a testdye. A 100-150 mg sample of each composition (from the 50-80 μm sizegroup) was first washed twice with methanol, allowed to dry, and thenimmersed in a saturated solution of 9-aminoacridine in methanol (10 wt%) for 24 hours. The methanol was allowed to evaporate (usually for 24hours) and the microspheres were washed by a repeated process ofsuspension and centrifuging (Adams Dynac centrifuge) in fresh neutral pHwater (at least 10 cycles). The loaded spheres were collected in a paperfilter cone and dried in an oven at 60° C. under vacuum.

For each composition, 5 mg of dye-loaded spheres were placed in glassvials (Fisher 20 ml scintillation vials) containing 15 ml of citric acidbuffer (0.05 M, I=0.3) at pH 6.0, 6.5, and 6.8. The buffer solutionsconsisted of citric acid (anhydrous, Fisher Scientific), NaOH (FisherScientific), deionized water, and NaCl (Aldrich). A total of threesamples were used from each composition at each pH value to account forerror involved with the weighing of the microspheres. At least fivesamples (5 ml each) were taken from each vial over a 14 day period andthe concentration of dye released was determined spectrophotometricallyat 400 nm using a Perkin-Elmer Lambda 3B UV/VIS spectrophotometer. Eachsample was replaced with fresh buffer solution. A 5 mg sample of eachcomposition was also placed into 15 ml of pH 2.5 citric acid buffer(0.05 M) for 24 hours to determine the total percent loading (mg dyereleased/mg loaded spheres) for the microspheres.

For each microsphere composition, the total percent loading (mg dye/mgmicrospheres) is shown in the following table:

TABLE 1 EMA/DEA/DVB 67/30/3 65/34/1 60/38.5/1.5 50/49/1 Wt % loading14.3 15.5 15.9 16.6

FIGS. 1-3 show the release of the test dye over a 14 day period at pH6.0, 6.5, and 6.8 for the 67/30/3, 65/34/1, and 60/38.5/1.5 ratios ofEMA/DEA/DVB microsphere compositions. In FIG. 1, the microspheres(67/30/3) showed complete release at a pH of 6.0 after 13 days, and lessthan 50% release at pH 6.5 and 6.8 after the full 14 days. At a pH of6.8 the spheres showed very little release after an initial burst whichmay suggest incomplete dye removal from the surface during the cleaningstage. FIGS. 2 and 3 show that by increasing the DEA content (as well asdecreasing the amount of crosslinking) of the spheres, an increase inthe overall release kinetics results. For the 60/38.5/1.5 composition ata pH of 6.8 there is still less than 50% of the dye released during thedesired two week period.

FIG. 4 shows dye release for the 50/49/1 composition at pH's of 6.5,6.8, 6.9, and 7.4. At a pH of 6.0 complete release was achieved over aperiod of several hours. At pH 6.8 and pH 6.9 about 76% and 63%,respectively, of the dye was released over the 14 day period. Theexperiment at pH 7.4 was done using a potassium phosphate monobasic-NaOHbuffer (0.05 M) (Fisher Scientific) to ensure that release was minimizedat a normal biological pH range. At this pH, roughly 14% of the dye wasreleased after two weeks. This release may be due to incomplete surfacecleaning or to release from a shallow surface layer with a shortdiffusion distance. Since the two week release range of this 50/49/1composition was near 6.7, it was chosen as the composition used for thein-vitro drug release experiments.

EXAMPLE 3 In Vitro Drug Release

During preparation for in vivo experiments it was determined that inorder to inject the microspheres into the hepatic artery of a rat, avery fine (26 gauge) needle was required Preliminary experiments showedthat the 50-80 μm diameter microspheres tended to clog the needle in asignificant percentage of initial trials. Since this was much less of aproblem for microspheres with diameters smaller than 50 μm a second setof release experiments was designed to determine what, if any, effectdifferent size ranges (35-50 μm and 80-150 μm) have on the loading andrelease characteristics of the spheres.

The in vitro drug release experiments were done using thechemotherapeutic drugs fluorodeoxyuridine (FUDR) (Sigma) and doxorubicin(hydroxydaunomycin hydrochloride) (Sigma). Due to the high cost andsmall quantities available for these two test drugs, the solutions usedfor loading the microspheres were limited to 5 mg/ml in methanol.

Initial FUDR Test. 150 mg of 50-80 μm diameter EMA/DEA/DVB (in a ratioof 50/49/1) microspheres were washed twice with methanol, dried and thenimmersed in 1 ml of methanol containing 5 mg FUDR. After 24 hours themethanol was allowed to evaporate and the loaded spheres were dispersedin 50 ml of distilled water and centrifuged repeatedly for 10 cycles.The spheres were collected and allowed to dry in a cone filter under thehood for 24 hours. 10 mg of the loaded spheres were placed in each oftwo separate vials containing either 15 ml of pH 6.8 citric acid buffer(0.05 M I=0.3) or pH 7.4 potassium phosphate monobasic-NaOH buffer (0.05M, Fisher Scientific). Each vial was agitated by hand daily and a totalof 5 samples (5 ml each) were taken over a 14 day period and measuredspectrophotometrically at 268 nm to determine the FUDR concentration insolution. Each sample was replaced using fresh buffer. The total percentloading was determined by the same process after placing a 10 mg sampleof spheres into 15 ml of pH 4.0 citric acid buffer for 24 hours.

Follow-up FUDR Release. After the initial FUDR release experiment showedthat the drug release was sufficient for measurement, the remainder ofthe FUDR release experiment was carried out as described in Example 2using citric acid buffer pH values of 6.6 and 6.9 with a total of threerepetitions for each. Sample volumes of 5 ml and 3 ml were taken fromthe pH 6.6 and 6.9 buffers, respectively.

Doxorubicin Release. The doxorubicin release experiments were carriedout in the same manner as the FUDR and dye release. 150 mg of 50-80 μmdiameter microspheres were cleaned and immersed in 1 ml of methanolcontaining 5 mg of the drug for 24 hours and the methanol was allowed toevaporate slowly under the hood. The loaded microspheres were cleanedand dried and 10 mg samples were placed into 20 ml glass vials (FisherScientific). 15 ml of citric acid buffer was placed into each vial suchthat there were three samples each at pH values of 6.6, 6.75, and 6.9.Drug release at pH 7.4 was performed using 15 ml potassium phosphatebuffer added to each of the remaining three vials. Drug release wasmeasured spectrophotometrically at 479 nm.

Size Related Dye Release. 100 mg samples of 35-50,50-80, and 80-150 μmdiameter EMA/DEA/DVB (50/49/1) microspheres were cleaned and loaded with9-aminoacridine using the same procedure described in Example 2. Releasewas carried out using 5 mg samples of spheres immersed in 15 ml of pH6.5 and 6.7 citric acid buffer solutions for 14 days. A total of threerepetitions were done for each size range at each pH. Solution dyeconcentrations were measured spectrophotometrically at 400 nm.

Dosages. FUDR (fluorodeoxyuridine) is commonly administered to patientswith primary or secondary liver carcinomas by continuous regionalinfusion. Most of the drug appears to be anabolized toFUDR-monophosphate, the active metabolite of the drug which inhibitsthymidylate synthetase thereby interfering with the synthesis of DNA.When doses are administered rapidly, FUDR is catabolized to fluorouracilwhich has similar effects as FUDR and metabolites of which interruptnormal RNA production (Budavari et al., 1989). Both FUDR andfluorouracil are metabolized in the liver, but this is somewhat reducedwhen the drug is given by continuous infusion rather than singleinjections. A standard FUDR dose given to human patients by hepaticarterial infusion is about 0.1 to 0.6 mg/Kg/day (Trissel, 1994) whilethe dose given to the rat model used by Ward et al. (1992) was either 1mg/Kg/day systemically or 2 mg/Kg/day regionally for 7 days. If themicrospheres were loaded at 14 weight percent (mg drug/mg spheres) withFUDR, there would be enough drug within 50 mg of the spheres to releasethis same 2 mg/Kg/day for 14 days within a typical 250 gram rat. For atumor burden estimated at about 10% of the liver mass at treatmentstage, this works out to be around 0.7 mg of FUDR released specificallywithin the tumor tissue with the remaining 90% trapped in themicrospheres or released at very low, non-cytotoxic levels within thehealthy liver areas. However, since it is known that preferentialhepatic angiogenesis of tumor tissue should deliver more particles tothe tumor (Meade et al., 1987), this is actually a worst case scenario.

Due to the high cost of the FUDR, a saturated loading solution todetermine the true percent loading of the spheres was not prepared. The5 mg/ml solution resulted in a loading level of only 1.4 weightpercentage of loading. However, since 500 mg samples of FUDR arecommonly constituted in 5 ml sterile water (100 mg/ml), it may beassumed that the drug solubility is such that high loading levels can beobtained. For comparison, 100 mg of 9-aminoacridine dye per ml ofmethanol were used earlier to load about 15% of dye.

FIG. 5 shows the release curves for the FUDR in buffer at four differentpH values. At pH 6.8 the spheres had released 80% of the encapsulateddrug over the 14 day period as compared to a 76% release of the test dyeunder the same conditions. At pH 6.9 the 14 day release values for FUDRand the dye were 55% and 63%, respectively. In both cases, just over 10%of the microsphere contents were released when exposed to pH 7.4. Therelease rate and the total fraction of released contents seem to comparewell for both the dye and FUDR loaded spheres despite the fact that theFUDR was loaded into the spheres under relatively dilute conditions.

Doxorubicin (hydroxydaunomycin hydrochloride) is commercially availableas the hydrochloride salt. It is an antineoplastic antibiotic but it istoo cytotoxic to be used as an anti-infective agent. The exact mechanismof its anticancer activity is not well understood but some evidencesuggests that the drug forms a complex with DNA which inhibits both DNAsynthesis and DNA-dependent RNA synthesis by the resulting templatedisordering. Cells that are the most sensitive to doxorubicin are fromrapidly proliferating tissues such as those of normal bone marrow,gastrointestinal mucosa, and hair follicles (Budavari, et al., 1989).Doxorubicin is administered intravenously and commonly used in thetreatment of solid tumors including bladder carcinoma, breast carcinoma,ovarian carcinoma, gastric carcinoma, malignant lymphomas, and acutelymphoblastic and myeloblastic leukemias. Doxorubicin is rapidlymetabolized in a first pass effect through the liver by an aldo-ketoreductase enzyme which forms doxorubicinol, the metabolite with themajor antineoplastic activity. The resulting plasma concentrations ofdoxorubicin and its metabolites are prolonged due to absorption by cellsand binding to cellular components such as nucleic acids (Trissel,1994). The plasma half life concentrations of doxorubicin and itsmetabolites is 16.7 and 31.7 hours, respectively, and can be longer inpatients with impaired hepatic function. The drug is primarily excretedin bile and in feces with only 4-5% excreted in urine. The use ofdoxorubicin for liver cancer treatment has for the most part beenlimited to hepatocellular carcinoma; however, it has produced clinicallyimportant responses in combination with other chemotherapeutic agentsand/or surgery in the early stages of this form of the disease(Budavari, et al., 1989). A common adult dose of doxorubicin would be a60 to 75 mg/m² (skin area), intravenous injection once every 21 days,but other schedules require smaller injections (20-30 mg/m²) either onceweekly or for 3 to 4 successive days every few weeks (Trissel, 1994).

FIG. 6 shows the release profiles for doxorubicin in citric acid bufferat 4 different pH values. At pH 6.75 the spheres release 75% of the drugover the two week period which is close to the 80% release obtained forthe FUDR at pH 6.8. Neither of the drugs nor the test dye show more than80% release over a pH of 6.75 during the 14 day period. However, allthree show complete release in under one week at a pH of 6.5 to 6.6.This indicates that the microspheres have a relatively sharp swellingtransition. Under in vivo conditions where the tumor pH is higher than6.7, the clinician can administer glucose to achieve a lowerintratumoral pH or use a suitable microsphere composition of the presentinvention that has a swelling transition to release loaded substances ata higher pH level.

FIGS. 7 and 8 show release profiles for three different size microspheresamples at two different pH values. In FIG. 7 the curves virtuallyoverlap which shows that at pH 6.5 the microsphere swelling is such thatthe dye release has little or no dependence on the sphere size. However,in FIG. 8, which shows release at pH 6.7, the 80-150 μm diameter spheresshow a noticeably slower release with a 14 day total of only 86%compared to 98% for the smaller spheres. The smaller spheres have moresurface area in contact with the buffer solution as well as shorterdiffusion distances which most likely explains the faster release. At pH6.7, which is relatively close to the swelling transition for thisparticular microsphere composition, the release rate is slow enough thatthis effect can be seen. However, as the pH becomes more acidic, themicrospheres release quickly enough that the difference can no longer beseen. At pH 6.7 there is virtually no difference between the releaseprofiles of the 50-80 μm and 35-50 μm diameter spheres, but as the pHincreases to 6.8-7.0 it is possible that the smaller spheres may show aslightly faster release rate.

EXAMPLE 4 In Vivo Compatibility

Empty Microsphere Injection. 200 mg of 50-80 μm diameter EMA/DEA/DVB(50/49/1) microspheres were washed by stirring in ethanol for 12 hoursand then dried under the hood before in vivo injection. The animals usedin this study were BD-IX rats weighing 150-250 grams (7-8 weeks old).All surgical procedures were performed under general anesthesia withphenobarbital injected intraperitoneally using 60 mg/Kg (body weight).

A midline incision was made in a rat, and with the aid of an operatingmicroscope the hepatic artery was isolated and ligated distally with a6-0 silk tie. This, along with a second loop placed proximally, was usedto control bleeding during the injection procedure. A few drops oflidocaine were applied to the artery to prevent spasms during theprocedure. A small incision was made in the artery using microdissection scissors and a catheter made from Biolab (0.023 inch innerdia.) vinyl tubing was inserted into the artery and secured by a silktie. A 25 gauge syringe needle was inserted into the catheter and asuspension (1 ml) made from 50 mg of the microspheres in a 50% glycerolaqueous solution was ultrasonicated for roughly one minute to break upany microsphere aggregates and then slowly injected into the artery overa 30 second period. Finally, 5 ml of sterile saline and 100,000 units ofpenicillin were injected into the peritoneal cavity and the incision wasclosed using a 4-0 silk suture. The skin was closed with 9-mm stainlesssteel autoclips and the rat was allowed to recover under warming lights.

Histology Slide Preparation. After 24 hours the rat was sacrificed andthe liver was harvested and fixed in 3% buffered formalin. The liver wasthen embedded in paraffin and cut into 5 μm sections using a microtome.The sections were placed on slides and the paraffin was removed usingacetone. The sections were finally re-hydrated with severalalcohol/water solutions with increasing water content. In order topromote microsphere visibility in the slides, the spheres were washedwith eosin (a basophilic stain) prior to the injection procedure.

Dye Loading and Injection. 500 mg of 50-80 μm diameter EMA/DEA/DVB(50/49/1) microspheres were cleaned by stirring in 40 ml ethanol for 12hours before being collected and dried under the hood. The spheres wereplaced into 3 ml of methanol containing 10 wt % 9-aminoacridine for 24hours. The loaded spheres were washed by 10 cycles of shaking in 50 mlof distilled water and then collected by centrifuging. The spheres werethen dried and separated using a spatula. The surgical procedure wasbasically the same as described previously except that it was performedon two separate rats which each received 150 mg of the microspheresinjected directly into the portal vein through a 23 gauge needle withoutthe use of a catheter. Rats were sacrificed immediately after injectionand their livers were removed and frozen.

Microsphere Retrieval. The removed rat livers were thawed and cut intosmall cubes roughly 1 cm wide. Each liver was put into 100 ml of a 2Mpotassium hydroxide (Fisher Scientific) solution and stirredmagnetically for 24 hours on low heat (45-50° C.). At this point theliver tissue was completely dissolved and the contents of each beakerwas poured 25 ml at a time into a 50 ml centrifuge tube (FisherScientific) with 25 ml of distilled water. Each tube was centrifuged for5 minutes and the supernate was removed. This was repeated until all ofthe dissolved liver solution had been used. The microspheres that werecollected were washed twice with water, dried in a paper cone filter(12.5 cm Whatman filter paper) under the hood, and then weighed. Two 5mg microsphere samples from each liver were placed into 15 ml of pH 4citric acid buffer solution for 24 hours. Also two 5 mg samples weretaken from the remaining microspheres that were not injected, and placedinto 15 ml of pH 4 citric acid buffer for 24 hours to serve as a controlgroup. The total dye release for each sample was measured at 400 nm byUV/VIS spectrophotometry.

Results—Dye Loaded Microsphere Retrieval. Because of the problemsassociated with the hepatic arterial injection of the microspheres, theportal vein was chosen in this experiment due to its larger size. Thisenabled the use of a larger needle (23 gauge) without the catheter whichgreatly reduced the chances of being blocked and allowed for a largerinjection volume of spheres (150 mg).

After 24 hours in a 2M potassium hydroxide solution the liver tissue wasdissolved leaving only some cellular debris remaining. Uponcentrifuging, the microspheres, which are more dense than water, formedthe bottom layer in the centrifuge tube. The majority of the cellulardebris remained suspended in solution, but there was a thin layer on topof the microspheres that had to be removed with a glass pipette (FisherScientific). A total of 77 mg and 91 mg of microspheres were removedfrom each of the two livers which were originally injected with 150 mg.Although some of the microspheres may have passed through the liverduring the injection process due to the broad size distribution, themajority of microspheres were most likely lost during the collectionprocess. Some of this probably could have been avoided if volumes largerthan 25 ml of the solution were centrifuged at a time because each timethe layers of debris were removed a small quantity of spheres were lost.

The loading percentages for the spheres collected from the livers were15.3% and 14.9% (weight of dye/weight of spheres) compared to thecontrol group which was loaded at 16.2%. Since the potassium hydroxidesolution is basic it is unlikely that dye was released during the tissuedissolution process. A trial experiment also showed that the spheres donot release in a 50% glycerol aqueous solution. However, during thesonication process it seems possible that any dye left on the surfaceafter washing could have been removed. The spheres were in living tissuefor only a short time (10-15 minutes) before being frozen so any releaseduring that time should be at a minimum. One possible reason for somedye release would be the two final washings with distilled water whichdoes have a pH of less than 7.0. Washings of the loaded spheres shouldbe done in a sodium hydroxide aqueous solution to reduce this effect. Itis also possible that a small percentage of the microsphere sample thatwas weighed after the procedure was actually left over cellular debristhat was not completely removed during the collection and washing of thespheres. Since this would result in a smaller quantity of spheres in thesample, less release would be expected. Overall, this experiment showedthat for a portal vein injection of microspheres, over 50% of theinjected spheres could be retrieved from the liver with at least 90% oftheir contents still intact. The collection procedure would be moredifficult for a hepatic arterial injection because of both the smallersize and quantity of the microspheres used; however, the use of smaller,more monodisperse microspheres should be effective.

EXAMPLE 5 Microsphere Size Control and Production ofMicrospheres—Dispersion Polymerization and Activated Swelling

Dispersion Polymerization. The dispersion polymerization experimentswere done using 25 ml glass vials with screw on caps (Fisher Scientific)placed horizontally in a constant temperature shaker bath (Blue M, MagniWhirl) set at 55° C. The standard components for each reaction weremethanol, poly(vinyl pyrrolidone) (PVP 40, 40,000 MW, Sigma),AIBN(re-crystallized in methanol), ethyl methacrylate,Diethyl(aminoethyl methacrylate), divinyl benzene, and ethylene glycoldimethacrylate (Aldrich). All of the following percentages are given inweight percent according to a total 10 gram sample.

The initial recipe for the polymerization reaction was chosen to be 10%monomer (3:2 ratio of DEA:EMA by weight), 3% poly(vinyl pyrrolidone)(PVP), 0.3% AIBN, and 86.7% methanol. The PVP was dissolved in themethanol and then filtered directly into the 25 ml reaction vial using a0.45 μm pore size (Whatman) syringe filter attached to a 10 cc syringe(Becton-Dickinson). The AIBN was dissolved into the monomer which wasthen mixed into the reaction solution by vortexing for 1 minute.Nitrogen or argon gas was bubbled through the reaction solution for atleast 2 minutes and the reaction vial was capped, sealed with PARAFILM(American National Can), and placed horizontally into the shaker bath at55° C. for 24 hours. This reaction was repeated 3 additional timesreplacing the 86.7% solvent portion with water/methanol ratios of 15/85,30/0, and 40/60. After each reaction was completed the solution waspoured into 100 ml of water and stirred magnetically for 1 hour, atwhich point the microspheres were collected by centrifuging and dried ina paper filter.

In order to determine the effect of initiator concentration on themicrosphere size, additional reactions were done using 0.1% and 0.4%AIBN. Both reaction solutions contained 10% monomer and 3% PVP with theremainder of the 10 gram total comprised of distilled water and methanol(30/70 wt %). The reactions were carried out under the same conditions.

The amount of PVP stabilizer was changed in two reactions to 1% and 5%with a constant initiator concentration of 0.3%. The monomerconcentration and the water/methanol ratio remained at 10% and 30/70,respectively. The reactions were carried out under the same conditions.

Three additional reactions were carried out using monomer concentrationsof 5%, 15%, and 20% (3:2 ratio of DEA/EMA by weight). The concentrationsof PVP and AIBN were 3% and 0.3%, respectively, and the water/methanolratio remained at 30/70. The reaction conditions were the same.

In an attempt to produce crosslinked microspheres, two reactions werecarried out using 0.3% and 0.6% (based on monomer weight) of ethyleneglycol dimethacrylate. The other variables were 15% monomer, 0.3% AIBN,3% PVP, and the remainder distilled water and methanol in a 30/70 ratio.

Activated Swelling Method. In the first step (pre-swelling) of thisprocedure 25 mg of monodisperse 2-3 μm diameter poly(ethyl methacrylate)(PEMA) microspheres were sonicated (Sonica & Materials, Inc., VibraCell) for 30 seconds in 3 ml of distilled water in a 5 ml test tube(Fisher Scientific) to disperse the spheres in solution. This was addedto an emulsion of 10 ml distilled water, 30 mg sodium dodecylsulfate(SDS) (Sigma), and 125 mg of dibutyl phthalate (Fisher Scientific) whichwas sonicated again for 1-2 minutes. The entire solution was put into a50 ml round bottom flask and was stirred magnetically for 24 hours. Inthe second step another emulsion consisting of 10 ml distilled water, 2grams monomer (3:2 ratio of DEA/EMA by weight), 20 mg divinyl benzene(crosslinking monomer), 20 mg SDS, 200 mg PVP, and 30 mg each of theco-initiators ethyl 4-dimethylaminobenzoate (Acros) andd1-camphoroquinone (Acros) was sonicated for 1-2 minutes and added tothe solution of swollen spheres produced in the first step. The solutionwas purged with nitrogen for 5 minutes while being magnetically stirredat slow speed. Once the microspheres became swollen with monomer(usually about 1 hour) a variable light source was turned on using a 140volt Staco Energy Products Co. Variable Autotransformer (set at 50%) andplaced roughly 8 inches from the round bottom solution flask. After 2hours the light source was removed and the solution was stirred for anadditional 24 hours. The reaction contents were poured into a 200 mlbeaker containing 100 ml of warm isopropanol (LabChem, Inc.) and stirredfor 2 hours before collecting the microspheres by centrifuging in 50 mlcentrifuge tubes.

Results. The variables which are involved in determining the particlesize distribution for a typical suspension polymerization processinclude stirring speed, the volume ratio of the monomer to suspensionmedium, stabilizer concentration, and the viscosity of both phases. Themechanical homogenization step produces an inherent size particledistribution which is typically anywhere from 20 μm to 2 mm in diameter(Arshady, 1992). Procedures such as wet sedimentation, counter flowsettling, and counter flow centrifugation can be used for the sizeseparation and classification but these methods are relatively difficultto implement and the reliability can be relatively poor (Hosoya et al.,1993). The microspheres produced in this research were separated intosize groups using metal sieves having different size openings. Attemptswere made to separate out a 10-20 μm diameter sample using 10 and 20 μmspectra fiber woven meshes, but there was just not enough microsphereswithin this range produced by the suspension method. Emulsionpolymerization techniques are commonly used to produce monodisperseparticles, however they are typically in the nanometer size range whichis far too small for use in this study (Arshady, 1992).

The dispersion polymerization process has become increasingly important,not only because it allows for the production of monodispersemicrospheres within the micron range (2-20 μm), but also because of thesimplicity of the process and the wide variety of monomers that can bepolymerized (Arshady, 1992, Shen S., 1993). Generally, the particlesizes produced using this technique range from 1-10 μm in diameter.However, by manipulating the reaction variables (Lok et al., 1985)produced monodisperse polystyrene spheres 12 μm in diameter (Arshady,1992). The dispersion polymerization reaction starts out as ahomogeneous solution consisting of monomer, solvent, initiator, andstabilizer in which the medium is miscible with the monomer but not thepolymer. As the initiator decomposes free radicals form and grow in thecontinuous phase until they reach a critical chain length where theyprecipitate out and form nuclei. The nuclei are unstable and absorb thepolymeric stabilizer chains while aggregating with each other untilenough stabilizer is absorbed forming mature particles. This particleformation stage continues until there are enough mature particles formedto capture all the radicals and nuclei in the continuous phase (Shen S.,1993). The mature particles then capture oligo-radicals and nuclei whichwill continue to grow inside the particles or terminate with otherradicals. At the end of the polymerization reaction the nuclei formationstops due to either lack of monomer or radicals. To form monodisperseparticles the reaction must have a short particle formation stage(compared to the growth stage) and a growth stage that is free from theformation of new particles and the coalescence of existing particles(Shen et al., 1994).

Initial experiments designed to produce monodisperse microspheres of thecorrect 50/49/1 EMA/DEA/DVB copolymer using dispersion polymerizationtechniques were done using methanol for a solvent, AIBN as theinitiator, and PVP as the stabilizer. The concentrations of 86.7%, 0.3%,and 3.0% (based on a 10 gram total including 10% monomer) respectivelywere chosen based on previous studies using poly(methyl methacrylate)(Shen S. et al., 1993, Bulmus et al., 1996). However, in this study itwas shown that the copolymer that was formed during the polymerizationreaction remained soluble until the solvent portion of the reaction(86.7%) included at least 30% distilled water (70% methanol). Thespheres produced from this recipe are reasonably monodisperse with theaverage diameter around 4 μm which was determined by optical microscopy(using graduated eyepiece) and verified by a scanning electronmicroscope (SEM). As the percentage of water in the solvent portion ofthe reaction was increased to 40%, the average size of the microspheresdecreased (1-2 μm dia.) but remained monodisperse. As the water contentis increased, the polymer formed is less soluble and tends toprecipitate out at lower molecular weights, which produces more nucleiin the particle formation stage, resulting in smaller particles (Tuncelet al., 1994). Therefore, in this study the lowest water/methanol ratiothat allowed polymer to precipitate (30/70) was used for the remainderof the experiments.

In the next set of experiments the initiator concentration was variedfrom 0.1% to 0.4% with the other variables constant. As theconcentration was increased, the size and polydispersity of the spheresincrease as well. These results were similar to those obtained by ShenS. et al. (1993) for poly(methyl methacrylate) (PMMA) microspheres whichincreased in size from 3 to 8 μm in diameter over the same range ofinitiator concentration. They determined that by increasing theinitiator concentration, the radical concentration increases, whichleads to the formation of lower molecular weight chains that are moresoluble in the reaction medium. Since the higher molecular weight chainsprecipitate to form nuclei, there ends up being a smaller number ofmature particles formed which grow to be a larger size. However, sincethis also affects the length of the particle formation stage of thereaction, there exists a maximum concentration of initiator forproducing monodisperse particles under given conditions. Themicrospheres produced using 0.3% AIBN were considerably moremonodisperse than those produced using 0.4%, so the 0.3% concentrationwas used in the remainder of the experiments.

Shen et al. (1994) showed that both the stabilizer concentration andmolecular weight had an effect on the final size of particles producedby dispersion polymerization. They concluded that an increase in thestabilizer concentration or molecular weight increases the viscosity ofthe medium, as well as the physical rate of stabilizer adsorption whichboth have the effect of reducing the extent of nuclei aggregation,resulting in an overall decrease in particle size. However, in thisstudy as the PVP concentration was varied from 1-5%, the change inmicrosphere size was minimal and the sample with a 5% concentrationshowed an increase in size distribution very similar to that seen forthe increasing initiator concentration. The only molecular weight of thePVP available for use in these experiments was 40,000 which was at thelow end of what was used in the study by Shen et al. (1994). The lowmolecular weight may account for the minimal effect of the concentrationon the particle size, but this was not shown to be the case in otherstudies (Shen et al., 1994, Bulmus et al., 1996).

All of the previous experiments were done using 10% monomer in thepolymerization recipe. In order to determine the effect of monomerconcentration on particle size, a series of experiments were done using5%, 15%, and 20% monomer, with the other variables at their optimumvalue. The batch with 5% monomer produced monodisperse microspheres, butas expected their average size was smaller than those produced inprevious experiments. The 15% monomer sample was larger (5 μm averagedia.) but the distribution became somewhat broader. A further increasein monomer concentration to 20% only increased the size distributionwith no noticeable particle size increase. These results generally agreewith those obtained by Shen S. et al. (1993) who determined thatincreasing the monomer concentration increases both the initial solvencyof the medium and the length of the particle growth stage which both actto increase the overall particle size. Therefore, it was determined thatfor this copolymer system the optimum values for the dispersionpolymerization variables were 0.3% initiator, 3.0% stabilizer, 10-15%monomer, and the remainder solvent (water/methanol 30/70 ratio).

Since the maximum monodisperse particle size that could be obtained bythis method was roughly 4 to 5 μm in diameter, several techniques wereattempted in an effort to grow these particles to within the desired10-15 μm range. These “seeded polymerization” methods failed due todissolution or aggregation of the particles caused by the monomer addedto the system. Efforts to overcome the dissolution problem bycrosslinking the particles during the dispersion polymerization processwere also unsuccessful because even low concentrations (0.3-0.6 wt %based on monomer) caused severe particle flocculation.

The activated swelling principle was first demonstrated by Ugelstad etal. (1980) and has become the only method available which allowspreparation of extremely monodisperse, crosslinked particles within themicron range (2-20 μm) (Christensen et al, 1996). Typically, it isdifficult to swell polymer particles with monomer because the particlesusually only absorb 1-10 times their own volume. Therefore, a seededpolymerization process may require multiple swelling and polymerizationsteps in order to double the diameter of the original seed particleswhich would increase the volume by a factor of 8. Since each stepincreases the chances of secondary particle formation and particleagglomeration, this process can prove to be unreliable (Ugelstad et al.,1980). In the first step of the activated swelling method, a “swellingagent” consisting of a highly water insoluble compound of relatively lowmolecular weight is introduced into the monodisperse particles (1:1 to1:5 volume ratio) in the form of an emulsion. Once the particles arecompletely swollen, a second emulsion is added containing the slightlywater soluble vinyl and divinyl monomers (and initiator). The“activated” particles may absorb from 100 to more than 1000 times theirown volume of the compounds added in this second step. The swollenparticles can then be polymerized by increasing the temperature of thereaction. Since all of the ingredients are introduced into the particlesbefore polymerization, the result is a high degree of monodispersity andbatch reproducibility (Ugelstad et al., 1980).

Dibutyl phthalate was used as a swelling agent in this study to swellhighly monodisperse (2-3 μm dia.) poly(ethyl methacrylate) microspheresproduced by dispersion polymerization techniques. PEMA microspheres wereused as seed particles because of the high degree of particle uniformityobtainable for that system. The comonomers, including divinyl benzene asa crosslinking agent, were added in the second step but due to thedegree of water solubility of the DEA monomer, the swollen particlesonly remained stable for roughly 2 hours. By increasing the temperatureto carry out the polymerization, the kinetics of the swollen dropletdegradation increased and very non-uniform particles were produced.However, this problem was overcome by substituting a photoinitiatorsystem composed of ethyl 4-dimethylaminobenzoate and d1-camphoroquinonefor the AIBN. Since this system resulted in room temperaturepolymerization within an hour, the swollen particles produced in step 2of the activated swelling method could be polymerized in time to retaintheir shape and size. The PEMA seed particles account for less than 2%of the final microsphere volume and since the PEMA is soluble in hotisopropanol, most (if not all) of the seed can be removed during thecleaning procedure.

Preliminary loading and release studies using the microspheres producedby this technique showed that they could be swollen in methanol andloaded with the test dye (9-aminoacridine). In three separateexperiments, loading levels between 15% and 16% were obtained which isonly slightly lower than the 16.6% loaded into the spheres produced bysuspension polymerization. A small difference can probably be expecteddue to both the differences in the microspheres size range and themonomer volumes used in each production technique (60 grams compared to2 grams). Also, the monodisperse microspheres were produced using twiceas much crosslinking content (2%) which decreases the overall loadingpotential.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

References

Arshady, R. (1992) “Suspension, emulsion, and dispersion polymerization:A methodological survey,” Colloid & Polymer Science 270:717-732.

Bhattacharya, S., Novell J. R., Winslet, M. C., Hobbs, K. E. F. (1994)“Iodized oil in the treatment of hepatocellular carcinoma” Br. J. Surg.81:1563-71.

Braun, A. C. (1974) The Biology of Cancer, Addison-Wesley PublishingCompany.

Budavari, S., Oneil, J. M., Smith, A., Heckelman, P. E., (Eds.) (1989)The Merck Index: an encyclopedia of chemicals, drugs, and biologicals,(11^(th) ed.), Merck & Co., Inc.

Bulmus, V., Tuncel, A., Piskin, E. (1996) “Production ofpolymethylmethacrylate particles by dispersion polymerization in aqueousmedia with ceric ammonium nitrate,” J. Appl. Polym. Sci. 60:697-704.

Busch, H., (ed.) (1974) The Molecular Biology of Cancer. Academic Press.

Christensen, B. E., Myhr, M. H., Aune, O., Hagen, S., Herge, A.,Ugelstad, J. (1996) “Macroporous, monodisperse partiless and theirapplication an aqueous size exclusion chromatography of high molecularweight polysaccharides,” Carbohydrate Polymers 29:217-223.

Hafeli, U. O., Siobhan, M. S., Bersford, B. A., Sim, E. H., Macklis, R.M. (1994) “Magnetically directed poly(latic acid) Y(90)-microspheres:Novel agents for targeted intracavitary radiotherapy” J. Biomed. Mat.Res. 28:901-908.

Hosoya, R., Frechet, J. M. J. (1993) “Influence of the seed polymer onthe chromatographic properties of size monodisperse polymeric separationmedia prepared by a multi-step swelling and polymerization method, ” J.Polym. Sci. Part A: Polym. Chem. 31:2129-2141.

Jahde, E., Ragewsky, M. F. (1982) “Tumor-selective modification ofcellular microenvironment in vivo: effect of glucose infusion of the pHin normal and malignant rat tissues” Cancer Res. 42:1505-1512.

Kato, T., Nemoto, R., Mori, H., Takahashi, M., Tamakawa, Y., Harada, M.(1981) “Arterial chemoembolization with microencapsulated anticancerdrug” JAMA 245:1123-1127.

Kemeny, N., Carr, B., Civalleri, D., Hakansson, L., Lindberg, B.,Gunnorsson, K., Nilsson, B., Taguchi, T., Khayat, C., Aigner, K. R.,(eds) (1995) An Update on Regional Treatment of Liver Tumours, The Roleof Vascular Occlusion, (Sec. Ed.), Wells Medical Ltd.

Lin, D. Y., Liaw, Y. F., Lee, T. Y., Lai, C. M. (1988) “Heparic arterialembolization in patients with unresectable hepatocellular carcinoma: Arandomized controlled trial” Gastroentereology 94:453-456.

Lok, K. P., Ober, C. K. (1985) “Particle size control in dispersionpolymerization of polystyrene,” Can. J. Chem. 63:209-216.

Meade, V. M., Burton, M. A., Gray, B. N., Self, G. W. (1987)“Distribution of different sized microspheres in experimental hepatictumours,” Eur. J. Cancer Clin. Oncol. 23:37-41.

Muir, B. L., R. N., M. Sc. (1988) Pathophysiology, An introduction tothe Mechanisms of Disease, John Wiley & Sons.

Narayani, R., Panduranga, K. (1995) “pH-Responsive gelatin microspheresfor oral delivery of anticancer drug methotrexate” J. App. Polym. Sci.58:1761-1769.

Oppenheimer, S. B. (1985) Cancer, A Biological and ClinicalIntroduction, second edition, Jones and Bartlett Publishers, Inc.

Shen, C., Rao, P. V., Batich, C. D., Moorhead, J., Yan, J. (1994)“Stochastic modeling of controlled release frompoly-styrene-co-4-vinylpyridine microspheres,” J. Controlled Release32:139-146.

Shen, C., Sudol, E. D., El-Aaser, M. S. (1993) “Control of particle sizein dispersion polymerization of methyl methacrylate,” J. Polym. Sci.Part A: Polym. Chem. 31:1393-1402.

Shen, C., Sudol, E. D., El-Aaser, M. S. (1994) “Dispersionpolymerization of methyl methacrylate: Mechanism of particle formation,”J. Polym. Sci. Part A: Polym. Chem. 32:1087-1100.

Spenlehauer, G. Veillard, M., Benoit, J. P. (1986) “Formation andcharacterization of cisplatin loaded poly(d,l-lactide) microspheres forchemoembolization” J. Pharm. Sci. 75:750-755.

Trissel, L. A. (1994) Handbook on Injectable Drugs,(8^(th) ed.),American Society of Hospital Pharmacists, Inc.

Tuncel, A., Kahraman, R., Piskin, E. (1994) “Monosize polystyrenelatices carrying functional groups on their surfaces,” J. Appl. Polym.Sci. 51:1485-1498.

Uglestad, J., Mork, P. C. (1980) “Swelling of oligomer-polymerparticles. New methods of preparation of emulsions and polymerdispersions,” Adv. in Colloid and Interface Sci. 13:101-140.

Volk, T., Jahde, E., Fortmeyer, H. P., Glusenkamp, K. H., Rajewsky, M.F. (1993) “pH in human tumor xenografts: effect of intravenousadministration of glucose” Br. J. Cancer 68:492-500.

Ward, E. S., Sigurdson, E. R., Tremiterra, S., Lincer, R., Chapman, D.,Niedzwiecki, D. (1992) “Adjuvant chemotherapy for colorectal hepaticmetastases: role of route of administration and timing,” SurgicalOncology 1:87-95.

We claim:
 1. A method of treating a patient having a tumor, said methodcomprising administering to the patient an effective amount ofpH-sensitive microspheres, wherein said microspheres comprise across-linked polymer gel comprising ethyl methacrylate, diethylaminoethyl methacrylate, and divinyl benzene, wherein said microspherescontain a substance selected from the group consisting of cytotoxicagents, chemotherapeutic agents, and radionuclides, and wherein saidmicrospheres are capable of releasing said substance at a pH rangeassociated with the tumor microenvironment, over a period of days. 2.The method of claim 1, wherein said microspheres are administered to thepatient by injection in a blood vessel proximate to said tumor.
 3. Themethod of claim 1, wherein said tumor is a hepatic tumor.
 4. The methodof claim 1, wherein the mole percent ratio of said ethyl methacrylate insaid microspheres is in the range of about 50 to about 65, wherein themole percent ratio of said diethyl aminoethyl methacrylate in saidmicrospheres is in the range of about 34 to about 49, and wherein themole percent ratio of divinyl benzene in said microspheres is in therange of about 1 to about
 15. 5. The method of claim 4, wherein the molepercent ratio of said ethyl methacrylate in said microspheres is about50, wherein the mole percent ratio of said diethyl aminoethylmethacrylate in said microspheres is about 49, and wherein the molepercent ratio of said divinyl benzene in said microspheres is about 1.6. The method of claim 2, wherein said blood vessel is the hepaticartery.
 7. The method of claim 1, wherein said microspheres aremonodisperse.
 8. A method of treating a tumor comprising administeringpH-sensitive microspheres in a blood vessel suitable for carrying saidmicrospheres to said tumor, wherein said microspheres comprise across-linked polymer gel comprising ethyl methacrylate, diethylaminoethyl methacrylate, and divinyl benzene, wherein said microspherescontain a substance selected from the group consisting of cytotoxicagents, chemotherapeutic agents, and radionuclides, and wherein saidmicrospheres are capable of releasing said substance at a pre-determinedpH, over a period of days.
 9. The method of claim 8, wherein saidmicrospheres.
 10. A pH-sensitive microsphere comprising a cross-linkedpolymer gel comprising ethyl methacrylate, diethyl aminoethylmethacrylate, and divinyl benzene, wherein said pH-sensitive microsphereis loaded with a substance selected from the group consisting ofcytotoxic agents, chemotherapeutic agents, and radionuclides, andwherein said pH-sensitive microsphere is capable of effectivelyreleasing said substance at a predetermined pH, over a period of days.11. The microsphere of claim 10, wherein said microsphere effectivelyrelease said substance at a pH of between about 6.0 and about 7.4. 12.The microsphere of claim 10, wherein said microsphere effectivelyreleases said substance at a pH of between about 6.3 and about 7.0. 13.The microsphere of claim 10, wherein said microsphere effectivelyreleases said substance at a pH of between about 6.5 and about 7.0. 14.The microsphere of claim 10, wherein said 50% of said substance isreleased and about 50% of said substance remains to be released whensaid microsphere is exposed to a pH of about 6.8 for a period of 14days.
 15. The microsphere of claim 10, wherein the weight percentage ofsaid substance loaded in said microsphere is up to about 20%.
 16. Themicrosphere of claim 10, wherein the mole percent ratio of said ethylmethacrylate in said microsphere is in the range of about 50 to about65, wherein the mole percent ratio of said diethyl aminoethylmethacrylate in said microsphere is in the range of about 34 to about49, and wherein the mole percent ratio of said divinyl benzene in saidmicrosphere is in the range of about 1 to about
 15. 17. The microsphereof claim 16, wherein the mole percent ratio of said ethyl methacrylatein said microsphere is about 50, wherein the mole percent ratio of saiddiethyl aminoethyl methacrylate in said microsphere is about 49, andwherein the mole percent ratio of said divinyl benzene in saidmicrosphere is about
 1. 18. The microsphere of claim 10, wherein saidmicrosphere has a diameter of between about 4 μm and about 150 μm. 19.The microsphere of claim 10, wherein said microsphere has a diameter ofbetween about 10 μm and about 50 μm.
 20. The microsphere of claim 10,wherein said microsphere has a diameter of between about 25 μm and about35 μm.
 21. A composition comprising a plurality of monodispersepH-sensitive microspheres, wherein said pH-sensitive microspherescomprise a cross-linked polymer gel comprising ethyl methacrylate,diethyl aminoethyl methacrylate, and divinyl benzene, wherein saidpH-sensitive microspheres are loaded with a substance selected from thegroup consisting of cytotoxic agents, chemotherapeutic agents, andradionuclides, and wherein said pH-sensitive microspheres are capable ofeffectively releasing said substance at a predetermined pH, over aperiod of days.